Oligo Microarray Probe Design

Question

[Eric Normandeau]

Hi all,

I'm in the process of designing a few thousands of oligo-nucleotide probes (60 pb) to construct an oligo-microarray.

My material consists of contigs assembled from a few runs of 454 cDNA sequencing as well as ESTs for the species I am working with. For the moment, I have extracted the sequences of interest and already trimmed them according to it's best blast region onto it's best protein equivalent in either swissprot or nr. I have also put them in the appropriate sense.

What I now need is a software/package/function to design 60 pb probes for these few thousand sequences. The tool must accordingly be scalable and if possible take into account any limitations/special issues linked to oligo-microarray probe design (eg: specificity of the probe to the target sequence, given a background group of sequences available for the same or other species).

What tool would you suggest and why?

Many thanks

[Paulo Nuin]

just a correction in your question, for microarray oligos are called probes.

[Anuraj Nayarisseri]

Premier Biosoft International Developed a tool called Alleleid or Beacon Designer. Try out this. This can help you out.

Answer 1

[Paulo Nuin]

My post-doc ex-supervisor has a software to generate probesets for gene families. Should work on your case too, and she released a parallel version (I did a simple interface). Check here

[Eric Normandeau]

Many thanks @Paulo! I tried your (or rather your collegue's) tool (command line version) and it works superbly :) Now I'll read the paper!

[Paulo Nuin]

It's a pretty good software, I recommend it. Also fast, you might need to generate some input files initially but it should be good to go. Unfortunately, I'm too far removed from it to help with details and features, but let me know if you have any question.

Answer 2

[Ketil]

You should also check with the micro array supplier, e.g. Agilent provides its own oligo design via its "eArray" web interface. Although the web application is rather clunky, designing oligos is just a matter of uploading the correct files (transcripts of interest, and complete transcriptome for cross-hybridization check).

[Ketil]

I guess Agilent won't thank me for suggesting it, but you can still register at eArray, and use it to design your probes. Which you can then download and use on your home-grown array :-)

[Eric Normandeau]

@Ketil: Thanks for the answer. However, we were building a custom microarray and where trying to avoid Agilent because of the costs. Cheers.

Answer 3

[Stefano Berri]

To design primer I always used primer3. A probe is not quite a primer, but they share many "properties". There is a web interface, but you can download the executable and run it at the command line or wrap it into your script. It is very mature and let you set a lot of parameters (annealing temperature, range of GC content, masking regions, ...)

[Eric Normandeau]

Hi @Stephano. Thank you for your answer. It is true that microarray probes and PCR primers share a lot. Both are shortish sequences with common properties (CG content, melting temperature...). However, Designing thousands of probes for an oligo-microarray also poses particular challenges. For one, specificity is very important. Probes should hybridize almost exclusively with the appropriate gene's cDNA. Designing probes one by one with primer3 is not going to guaranty against non-specificity or even try to reduce it. I'd be glad to hear your thoughts or suggestions! Cheers.

Answer 4

[Puthier]

Did you test OligoWiz?

[Eric Normandeau]

Negative, but I just added the link to my online ressources. Maybe for a next project. However, I have been really pleased by the program suggested by Paulo Nuin (see accepted answer). Cheers!

[Puthier]

Ok. I will have a look at ProDesign also. Thanks

Answer 5

[Anuraj Nayarisseri]

Premier Biosoft International has developed a tool called Allelid. They have one more tool called Beacon Designer. Both these tools can help you out to design Microarray probes. Here is the link.

Bowang

Answer 6

[Sean Davis]

It really isn't too hard to do this by brute force. For example, design all possible 60mers for all targets and then use blat to determine the uniqueness to the level of interest (best hit to target and no more than 50% match to off-target, etc.). Compute %GC and Tm and exclude stuff too far off from your goal. If you want to get fancy, you can check for secondary structure as well.